Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The ability to isolate viral DNA and to clone this DNA into bacterial plasmids has greatly expanded the approaches that can be used to make viral vaccines. The approach in this application is to modify the viral DNA sequence while in the cloned state in a plasmid, modifications which include, but are not limited to, insertion, deletion, or single or multiple base change. The modified DNA is then inserted back into the viral genome for the purpose of rendering the virus nonpathogenic. The resulting live virus product can be used to elicit an immune response and be protective as a vaccine, or in any other situation wherein a nonpathogenic virus infection of an animal is required.
One group of animal viruses, the herpesviruses or herpetoviridae, is an example of a class of viruses amenable to this approach. These viruses contain 100,000 to 150,000 base pairs of DNA as their genetic material, and several areas of the genome have been identified that are dispensible for the replication of virus in vitro in cell culture. Modification of these regions of the DNA are known to lower the pathogenicity of (to attenuate) the virus for the animal species. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic (1), and pseudorabies virus of swine non-pathogenic (2) and (3).
Removal of part of the repeat region renders human herpes simplex virus non-pathogenic (4) and (5). A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity and changes in this region correlates with lack of oncogenicity (6). These modifications in the repeat region do not teach the construction of attenuated pseudorabies viruses with deletions in repeat sequences. A different region in herpesvirus saimiri has similarly been correlated with oncogenicity (7). A region in pseudorabies virus has been shown to be deleted in naturally occurring vaccine strains (8). This deletion is partly responsible for lack of pathogenicity, however it does not occur in a repeat sequence and does not suggest attenuation resulting from a deletion in a repeat sequence.
The general conclusion from the literature is that herpesviruses contain nonessential regions of DNA in various parts of the genome, and that modification of these regions can lead to attenuation of the virus and derivation of a vaccine or non-pathogenic strain. The degree of attenuation of the virus is important in the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response.
The herpesviruses are known to cause a variety of latent and recurrent infections in human and other vertebrates and are even known to infect a fungus and an oyster. Among the conditions associated with herpesvirus infection are fever blisters caused by herpes simplex type 1, genital herpes caused by herpes simplex type 2, and chickenpox in children and shingles in adults caused by herpes zoster infection. A Class D herpes virus known as pseudorabies virus (PRV) induces Aujeszky's disease, an acute and often fatal nervous condition, in domestic and wild animals.
The natural host of pseudorabies virus is swine, in which infection is commonly inapparent but may be characterized by fever, convulsions and paralysis. Pseudorabies virus also infects cattle, sheep, dogs, cats, foxes and mink, where infection usually results in death of the host. The predominant visible feature of pseudorabies virus infection is intense pruritis generally resulting in host mutilation of the involved area. Violent excitement, fits and paralysis, all symptoms of encephalomyelitis, precede death which usually occurs within a few days following onset.
The pseudorabies virus genome has been mapped (See FIG. 1). The genome is known to include, in order, a unique long region, an internal inverted repeat sequence, a unique short region, and a terminal inverted repeat sequence.
Pseudorabies virus disease in swine is of serious concern by governmental bodies worldwide. In the U.S., infected swine cannot be sold except to slaughterhouses. Several individual states have separately enacted eradication control practices against pseudorabies. The R & D trend among traditional vaccine manufacturers has been to emphasize research leading to products that are based upon the subunit vaccine approach rather than using live viruses. This departure from live virus vaccines is due mainly to the recognized safety aspect of subunit vaccines, and their unlikelihood of containing infectious live viruses. It is well known that traditional live virus vaccines can revert to virulence which would seem to maintain a source of the disease during an eradication program. Attenuated pseudorabies viruses which have a deletion in at least a portion of a repeat sequence are a departure from most current live virus vaccines, and from the direction in which the vaccine industry as a whole is moving. These viruses suffer deletions in the repeat region and in other regions which cannot be repaired, i.e. the viruses cannot revert to virulence and are safe. A vaccine comprised of these viruses combines the safety aspect of subunit vaccines with the commercial and efficacious advantages of live virus vaccines.
Vaccines against pseudorabies virus are available. These vaccines are generally produced by the technique of cell culture attenuation. It is known that some live virus vaccines produced by this method contain deletions in the unique short region of the viral genome (8), however these viruses do not have deletions in repeat sequences.
On May 15, 1985 European Patent Publication No. 0 141 458 was published. This European Patent Publication is based upon European Patent Application No. 84201474.8, filed on October 12, 1984, and is entitled "Deletion Mutant of a Herpesvirus and Vaccine Containing Same".
This publication discloses the construction of attenuated pseudorabies viruses which have sequences of DNA deleted from the genome. Among the types of deletions are deletions in the unique short region and deletions in the repeat sequences. However this application does not provide enough detail to teach attenuating deletions in repeat sequences nor does it suggest the unexpected attenuating nature of deletions in repeat sequences. Specifically the inventors state that a small deletion in the repeat unit at the position of the Hind III site is not of importance for virulence in pigs, thereby indicating directly that they were not aware of the importance of the repeat region in attenuation. In any event, this application was published in a foreign country less than one year before the filing date of this application.
It is also known that deletions in the thymidine kinase (Tk) gene of the pseudorabies virus, located in the unique long region of the genome, renders the virus non-pathogenic but reduces the immune response in the host animal as well. U.S. Pat. No. 4,514,497, entitled "Modified Live Pseudorabies Viruses", discloses temperature resistant pseudorabies viruses which have deletions in the TK gene only and does not teach or suggest pseudorabies viruses which are attenuated by deleting portions of repeat sequences. In addition our attenuated pseudorabies viruses have never been selected for any kind of temperature resistance.
An ideal live virus vaccine is non-pathogenic and produces a strong immune response. It is therefore desirable to produce a live pseudorabies virus vaccine which would be non-pathogenic and produce a strong immune response in host animals.
This invention concerns attenuated pseudorabies viruses, i.e. pseudorabies viruses with lowered pathogenicity, which are useful as a vaccine to elicit a strong immune response or in other situations wherein a nonpathogenic viral infection of an animal is required. These viruses have a deletion in at least a portion of a repeat sequence. When used to immunize animals against pseudorabies virus disease, these viruses provide the advantages of not killing foreign animals or being passed to companion animals. Additionally, the viruses are stable, i.e. they do not revert to a pathogenic strain, and they provide a good immune response.